Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate

Prior strategies to measure inositol 1,4,5-trisphosphate (IP3) in single ce lls either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IF, in small regions of a Xenopus oocyte, To improve th e detection limits of this method, we elucidated the experimental parameter s which influenced the sensitivity and reliability of the IP3-detector cell coupled to capillary electrophoresis, The variables which influenced the d etector cell were the magnitude of the voltage drop across the detector cel l, the duration of this voltage drop, the direction of fluid how in the cap illary, the concentration of free Ca2+ around the detector cell, and the pr esence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cel l acted primarily as an IF, mass detector rather than a concentration detec tor. Characterization of the experimental variables influencing the sensiti vity and reliability of this detector cell has the potential to enhance oth er analyte measurements performed by mating capillary electrophoresis with a biological detector cell. (C) 2000 Academic Press.